Industrial strains of bacteria are used in the production of many food products including yogurt, sour cream, cheese, sausage, and sauerkraut. Bacterial strains which exhibit stable characteristics are essential to maintaining the flavor of the food product, the texture of the food product, and the quality of food product produced. An example of a bacterial genus of importance to the food industry is Lactococcus, strains of which carry out dairy fermentations by fermentation of lactose and degradation of milk proteins.
The proteins and enzymes involved in the fermentation and degradative pathways, as well as other desirable traits, are often encoded on plasmids. In some bacterial strains, these plasmids are easily lost resulting in bacterial strains with unstable characteristics and inconsistency in the quality, flavor, and type of food product produced by the strain. This problem is exacerbated when food production is scaled up and loss of desireable traits or characteristics of the bacteria causes the loss of the desirable fermentative properties and formation of an inferior and/or unacceptable food product.
One way plasmid encoded genetic traits or characteristics can be stabilized is by integration of an integration vector like a plasmid or a virus into the chromosome of the bacteria. Integration of vector genetic material into the bacterial chromosome can occur by recombination of the vector genetic material at a homologous site on the bacterial chromosome in a single or double crossover event. A single crossover event results in integration of the entire vector. Double crossover events result in incorporation of a portion of the vector genetic material. Once the vector genetic material is integrated into the chromosome, it is replicated as a part of the chromosome and the associated traits can be stably maintained for numerous generations.
Integration vectors have been used as genetic tools in a number of different bacteria. Vectors have been designed to delete chromosomal genes, to exchange wild type genes with mutated genes generated in vitro, to map chromosomal genes, to determine complementation and dominance, to generate in vivo gene fusions for transcriptional studies, to insert genes into conjugal transposons for transfer to non-transformable strains, to clone chromosomal genes, and to stabilize essential genes in industrially important microorganisms. An example of the use of integration vectors in the stabilization of essential genes is the integration of .alpha.-amylase genes from one species of Bacillus into the chromosome of another species of Bacillus by a nonreplicating vector containing random Bacillus chromosome fragments as reported by P. Kallio et al. in Appl. Microbiol. Biotech., 27:64-71 (1987). The .alpha.-amylase gene was stably integrated, amplified and expressed in the Bacillus species.
The usefulness of integration vectors for transforming industrial bacterial strains depends upon the stability of the integrated sequences in the chromosome. The stability of the integrated plasmids is influenced by the type of crossover event which results in integration and on the type of vector. Vectors, such as RCR plasmids, whose retention of residual replication ability stimulate recombination and excision of the vector genetic material integrated by single crossover recombination, are not stably integrated. In contrast, non-RCR plasmids which integrate by single crossover events, with or without gene amplification, remain stably integrated in Bacillus as reported by L. Janniere et al. in Gene, 40:47-55 (1985).
Nonfood-grade vectors have been constructed, like for example, by insertion of antibiotic resistant marker genes and lactococcal chromosomal DNA into a non-lactococcal plasmid which cannot replicate in Lactococcus species. Several nonfood-grade lactococcal integration vectors have been studied, with varying abilities to provide stable integration into lactococcal species. K. Leenhouts et al., in Appl. Environ. Microbiol., 56:2726 (1990), describe derivatives of plasmids from E. coli and Saphylococcus aureas which contain lactococcal chromosomal DNA and an erythromycin resistance gene that were capable of integrating into the chromosome of a strain of Lactococcus lactis. These results indicate great promise for stabilizing genes with integration vectors in bacterial strains of industrial importance.
In order for integration vectors to be currently applied in industrial processes to stabilize desireable genetic traits, the integration vector must be a food-grade vector. Food-grade vectors are considered acceptable for use in bacteria to be consumed by humans. To be food grade, the vectors are constructed of DNA corresponding to DNA sequences derived from microorganisms used in food and dairy fermentations, do not require passage through another microorganism not used in food or dairy fermentation, and contain a food-grade selectable marker gene. As described previously, currently available integration vectors are not food-grade because either they contain DNA from another nonfood related genus of bacteria or they must be passed or amplified in another genus of bacteria or they lack a food-grade selectable marker gene. The two major obstacles for development of stable food-grade integration vectors have been the lack of easily selectable food-grade marker genes and the lack of conditionally maintained plasmids from which to construct integration vectors.
Accordingly, there is a need to develop food-grade integration vectors containing easily selectable food-grade marker genes. There is also a need to identify vectors which are composed of genetic material from strains of industrial bacteria to serve as food-grade integration vectors and which do not require passage through another type of bacterial host. There is also a need to develop industrial strains of bacteria that have stably integrated industrially valuable characteristics or traits, including fermentation of lactose and degradation of milk proteins.